• Clinical and Experimental Reproductive Medicine
• /
• 제26권1호
• /
• pp.75-81
• /
• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• 한국자원식물학회:학술대회논문집
• /
• 한국자원식물학회 2000년도 춘계임시총회 및 학술발표대회
• /
• pp.69-71
• /
• 2000

• 한국동물학회:학술대회논문집
• /
• 한국동물학회 1996년도 공동 춘계학술대회
• /
• pp.56.2-56
• /
• 1996

No Abstract, See Full Text

• Reproductive and Developmental Biology
• /
• 제36권4호
• /
• pp.269-273
• /
• 2012

The present study was to assess the in vitro viability and sexing rate of bovine embryos. Blastocysts were harvested on day 7~9 day after insemination(in vitro and in vivo), and the sex of the embryos was examined using the LAMP method. Embryo cell biopsy was carried out in a $80{\mu}l$ drop $Ca^{2+}$, $Mg^{2+}$ free D-PBS and, biopsied embryos viability were evaluated after more 12 h culture in IVMD culture medium. The formation of recovered embryo to expanded and hatching stages had ensued in higher of sexed embryo in vivo than in vitro (100% vs. 89%, p<0.05), and in vitro, the rates of degeneration after sexing were significantly (p<0.05) higher in vitro than in vivo(11% vs. 0.0%). The rates of the predicted sex were female 61% vs. 56%, and male 39% vs. 44% in vivo and in vitro, respectively. The rates of survival following different biopsy methods were seen between punching and bisection method in vivo and in vitro (100% vs. 89% and 100% vs, 78% respectively). Biopsy method by punching was significantly (p<0.05) higher than bisection between produced embryos in vivo and in vitro. The present data indicate that with microblade after punching for embryo sexing results in high incidence of survivability on development after embryo biopsy. It is also suggested that LAMP-based embryo sexing suitable for field applications.

• 한국수정란이식학회지
• /
• 제31권2호
• /
• pp.123-129
• /
• 2016

This study is performed to evaluate the effect of insulin in the porcine parthenogenetic embryo development. In porcine embryo culture, insulin is helpful factor in the process of embryo development. To identify this, insulin is used in pig embryos development. Therefore, this study was performed to investigate the effect of insulin on early embryonic development in pigs. For that, insulin positive or negative (0, 10 ug/mL) was supplemented in the porcine IVM media and then compared two groups divided by the cytoplasm of the black groups and white ring groups based on the distribution of lipid material of the cell cytoplasm in microscope. In maturation rates of porcine oocytes, significant higher black group rates were shown in the insulin positive groups compared with other groups ($56.0{\pm}2.1$ vs $46.2{\pm}0.3$). In the embryo culture, black groups were showed the significant higher cleavage rates ($82.1{\pm}0.8$, $78.3{\pm}0.1$ vs $63.2{\pm}0.3$, $63.4{\pm}0.0$), and blastocyst formation rates ($15.5{\pm}3.6$, $16.6{\pm}0.4$ vs $11.7{\pm}1.3$, $7.4{\pm}0.2$) regardless of whether the addition of insulin. Also, black groups were showed higher cell number of blastocyst ($33.2{\pm}2.5$, $35.5{\pm}2.6$ vs $31.2{\pm}2.1$, $31.3{\pm}2.2$). In conclusion, supplement of insulin producing black group in vitro maturation, it was effective in vitro maturation and embryonic development of pig embryos.

• 한국수정란이식학회지
• /
• 제33권3호
• /
• pp.139-147
• /
• 2018

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for animal cloning. Until now, SCNT has been inefficient, especially in dog. It is believed that an embryo developmental block in SCNT embryos is cause of low production efficiency. However, no studies have been performed on canines for embryo developmental block. In this study, we attempted to evaluate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into 0.01 mM EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, Reactive Oxygen Species (ROS) activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Furthermore, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Moreover, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, we elucidated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

• Clinical and Experimental Reproductive Medicine
• /
• 제28권1호
• /
• pp.55-63
• /
• 2001

Objectives: This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Materials and Methods: Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. Results: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1 %,79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4-8 cell embryos in PROH were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The developmen1 rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. Conclusions: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH showed better development rates of embryos in 2 and 4-8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

• 생명과학회지
• /
• 제25권2호
• /
• pp.158-163
• /
• 2015

쌀의 쌀눈은 배유에 비해 단백질, 지방, 비타민 B1 등의 영양분을 더 많이 함유하고 있다. 본 연구에서는 식물에서 발현 할 수 있는 p53 플라스미드를 제작하였으며, 여러가지의 배지에서 쌀눈 발아의 최적조건을 확립하였다. p53 플라스미드는 pcDNA-p53 플라스미드에서 p53을 얻어내어 TA 벡터에 subcloning을 한 후 식물 플라스미드인 pGEM-CaMV에 p53을 삽입하여 식물에서 발현 가능한 pGEM-CaMV-p53 플라스미드를 제작하였다. 그리고 효율적인 p53 유전자의 도입을 위하여 최적의 팽윤버퍼의 조성 및 배지의 조건을 확립하였다. 팽윤방법을 통한 유전자의 도입에서 팽윤버퍼는 염과 detergent의 서로 다른 농도로 조성하였지만, 이 버퍼조성 사이에서의 쌀눈 발아율의 유의한 차이는 확인되지 않았다. 또한 팽윤된 쌀눈의 발아를 극대화 시키기 위하여 고체한천배지, 액체 배지, 페이퍼 타올 배지의 3가지 조건에 대해서 발아실험을 진행하였다. 그 결과 고체배지에서의 발아율은 70% 정도로 가장 높고 액체배지에서의 발아율은 20% 정도로 가장 낮았으며, 페이퍼 타올배지에서의 발아율은 60% 정도였다. 앞서 확인한 최적의 발아조건에서 쌀눈에 p53 플라스미드를 도입하였고, 그 결과 쌀눈에서의 human p53 발현을 확인 할 수 있었다. 따라서, 팽윤방법에 의한 쌀눈에서의 효율적인 유전자 발현은 쌀의 새로운 부가가 치를 창출할 수 있을 것이다.

• 한국수정란이식학회지
• /
• 제17권3호
• /
• pp.203-209
• /
• 2002

본 실험은 원시난포의 체외배양 체계를 확립할 수 있는 가능성을 검토하기 위해 10일과 20일령 마우스의 난소에서 분리한 난포를 체외배양하여 난포의 형태변화, 난자의 성장를 및 난자의 핵성숙단계를 조사하여 비교하였다. 그 결과를 요약하면 다음과 같다. 1. 10일령과 20일령의 마우스에서 분리한 난포는 배양 6일째부터 난포강이 형성되었다. 2. 10일령과 20일령 마우스의 난소에서 분리한 난포의 수는 평균 21.5와 33.3개였고, 배양하기 전에 투명대를 제외한 난자의 직경은 51.85 $mu extrm{m}$와 57.50 $\mu\textrm{m}$였으나, 10 일간 배양한 후 측정한 난자의 직경은 55.95 $\mu\textrm{m}$와 63.11 $\mu\textrm{m}$로 증가하였다. 3. 10일령과 20일령에서 M II까지 핵이 성숙된 난자는 각각 4.3%와 22.1%였고. GVBD기 이상은 각각 14.5%와 61.1%였다.

• 한국가축번식학회지
• /
• 제18권2호
• /
• pp.113-119
• /
• 1994

To improve nuclear transplantation(NT) efficiency and to produce a large scale genetically identical cloned calves, examined the in vitro development capacity after co-culture of bovine oviductal epithelial cells (BOEC) and granulosa cells in TCM-199 supplemented with 10% fetal calf serum (FCS) with early bovine embryos derived from in vitro matured fertilized(IVM-IVF) oocyte. In addition, the age dependence of IVM oocyte on electro-stimulation and the effective electric voltage on in ivtro development of bovine NT embryos were examined. The results obtained were summerized as follows; 1. The cleavage rates of IVM-IVF bovine embryos in co-culture with bovine oviductal epithelial cells and granulosa cells were not significantly different(P<0.05), but the developmental rate into morula and blastocyst stage were different showing 38.3 and 20.2%, respectively. 2. The activation (82.5%) and development in vitro(8.6%) into later embryo stages of the aging oocytes of 32 hours post-maturation (hpm) were significantly higher than those of 24 hpm at direct current (DC) voltage of 1.5kV/cm, 60$\mu$sec pulse duration and 1 pulse time. 3. The fusion rates of NT eggs of 32 hpm following to different DC voltages from range 0.75 to 1.5kV/cm were not differ, but the developmental rate into morula and blastocyst stages at DC voltages of 0.75 and 1.0kV/cm were higher(11.4 and 12.6%, respectively) than those of 1.5kV/cm(0%). From these results, it can be suggested the optimal culture system for in vitro culture of IVM-IVF bovine embryos is a co-culture system with BOEC in TCM-199 supplemented 10% FCS. The effective time and the DC voltage for activation, electrofusion and in vitro development of NT embryos derived from IVM-IVF bovine embryo are 32hpm and 0.75~1.0kV/cm. But to improve NT efficiency, the advanced research (cell cycle synchronization, micromanipulation, culture system, etc.) is needed.